1. Field of the Invention
The present invention relates to a method of screening for inflammatory diseases in a patient. More particularly, this invention concerns the method of screening a patient for the presence of inflammatory diseases such as an intraamniotic infection, bacterial meningitis and the sexually transmitted diseases; gonorrhea, chlamydia and trichomoniasis, by utilizing neutrophil defensins and lactoferrin, found in a bodily fluid, a tissue or a combination thereof, to give an indication of whether the patient is at risk of suffering from one or more such inflammatory diseases.
2. Description of Related Art
The methods of disease detection may be divided into two general types: diagnosis and screening. Diagnosis is the method whereby a physician determines the nature of a disease based upon the patient's signs and symptoms. Screening is the method of suggesting the presence, or the absence, of a particular disease, or class of diseases, in a patient. When a screening test indicates that a patient does not have a disease, in many cases the need for further diagnostic testing has been eliminated. Used in this manner, screening saves money for patients, health insurance companies and government health programs by precluding the unwarranted diagnostic testing of people shown not to suffer from the disease or class of diseases. To be effective in reducing unnecessary diagnostic testing, however, a screening method must be widely used. In order that a screening method is widely used, the screening test should be relatively accurate, quick, and economical to use.
In addition, screening provides a way for patients to avoid the cost and discomfort associated with the more invasive procedures often necessary to collect the samples required for diagnostic testing. The following are some of the conventional methods used for screening and diagnosing inflammatory conditions such as an intraamniotic infection, bacterial meningitis, and the sexually transmitted diseases; gonorrhea, chlamydia and trichomoniasis.
a. Intraamniotic Infections
An intraamniotic infection is an infection of the amnion or of the amniotic fluid by any pathogen. It is thought to be a significant cause of idiopathic preterm labor, which results in preterm deliveries. The typical screening procedure for an intraamniotic infection involves the physician empirically identifying women in preterm labor. Unfortunately, the majority of women with preterm labor do not have overt signs or symptoms of intraamniotic infection, such as fever, increased heart rate and abdominal tenderness between contractions.
The standard test for diagnosing an intraamniotic infection involves the physician obtaining an amniotic fluid sample from the patient via amniocentesis. This is followed by growth of a culture from the extracted fluid. Diagnosis is both costly and time consuming, because a physician must perform the amniocentesis procedure to procure the necessary sample and skilled laboratory personnel are needed to grow and analyze the culture. Growth of the specimen requires at least 24 hours, which significantly delays treatment. Further, diagnosis is often negative in cases where there is significant placental infection or inflammation. Also, the procedure itself can present a risk to the fetus, and discomfort to the mother. Therefore, many clinicians diagnose intraamniotic infections solely based on the inability to stop labor with tocolytics. Recent evidence linking infection to cerebral palsy suggests that this practice may potentially be harmful. Clearly, more rapid means of identifying patients with intraamniotic infection are needed.
b. Bacterial Meningitis
Bacterial meningitis afflicts approximately 10,000 people annually. The typical screening method for bacterial meningitis involves empirical observation by the physician of the presence of the following symptoms in children: fever with temperature, instability, 35 irritability or lethargy, refusal to feed, vomiting and diarrhea and respiratory distress. In adults, the physician looks for: fever, headache, meningismus or irritation of the lining of the brain and altered mental status. Complications of bacterial meningitis can include death and neurologic sequalae in approximately 10-20% of patients. Figures are higher in immunosuppressed patients such as newborn babies. Fortunately, early diagnosis and treatment will reduce these complications.
Bacterial meningitis is typically diagnosed by first obtaining a sample of cerebrospinal fluid (CSF) from the patient by means of a lumbar puncture. This sample is then cultured and stained for Gram positive bacteria. Also, measurements of white blood cells, glucose and protein in the cerebrospinal fluid can be made. Deviations in these parameters from normal levels can be used by the physician to diagnose bacterial meningitis. A CSF white blood cell count greater than 1000/.mu.L, a CSF glucose level less than 30 mg/dL and a CSF protein level greater than 100 mg/dL, can be used alone or in combination to diagnose bacterial meningitis in a patient. The major drawback to using these tests individually to diagnose bacterial meningitis lies in their low sensitivity. While positive Gram stains have an eighty-four percent (84%) sensitivity, white blood cell counts have a sensitivity of only fifty-three percent (53%), and glucose levels have only a fifty-eight percent (58%) sensitivity. Protein levels have a sensitivity of ninety percent (90%). Therefore, these tests are typically utilized in combination, with an abnormality in any one test being used to predict bacterial meningitis. If all four tests are used in this manner, they can result in approximately a ninety-five percent (95%) sensitivity. (See Table 2) Performing all these tests, however, can also result in more expense being borne by the patient because a physician must perform the lumbar puncture and a competent laboratory must conduct the testing. Making a diagnosis in this manner can also result in a longer time being required because the physician may have to await the results from four tests before being certain that there is an abnormality in at least one test.
c. Sexually Transmitted Diseases
The typical screening method for sexually transmitted diseases involves the physician noting the patient complaining of painful or difficult urination, called dysuria, and abnormal discharge. The physician then checks the patient for redness, swelling or sores on or about the genitalia. Additional screening methods include using the leukocyte esterase dipstick or neutrophil quantification on Gram stain or wet smear. Unfortunately, these methods have sensitivities of thirty-three percent (33%) to eighty percent (80%) with specificities of fifty percent (50) to eighty percent (80%) thereby making them less than ideal for routine clinical use.
Another type of screening method for sexually transmitted diseases, employed by the World Health Organization and by some Third World countries, involves the use of an algorithm comprising a series of questions. Because this screening method is based solely on the patient's answers to questions, which may be either inaccurate or untruthful, and not on any physical assessment of the patient by a physician, the degree of error is likely to be quite high. Further, because of the scarcity of resources in Third World countries, no other test is performed in those cases where the answers do not suggest the presence of an infection, thereby neglecting many patients who are infected.
Several diagnostic tests for sexually transmitted diseases are readily available in the United States and other developed nations. The most accurate diagnostic techniques are PCR and LCR, both of which amplify the amount of pathogenic microbial genetic material in a patient specimen to detectable levels. The use of PCR and LCR is limited by the expense involved in having a physician collect a specimen from the patient and the costs for a proficient facility to perform the necessary lab work.
(1) Gonorrhea
Gonorrhea is caused by the gonococcal bacterium Neisseria gonorrhea. If undetected and therefore untreated, gonorrhea can cause postgonococcal nonspecific urethritis, epididymitis, pelvic inflammatory disease, arthritis and even death. There were one million (1,000,000) cases of gonorrhea reported in the United States and approximately one-hundred- twenty million (120,000,000) cases worldwide. One method commonly employed in diagnosing gonorrhea involves performing a Gram-stained smear on a scraping taken from the patient. A culture is required for females because a Gram-stain is considered less reliable for them. There are several disadvantages to the use of this diagnostic method. First, it is costly because it requires a physician take the sample required for the test. Second, it is time consuming because a culture must be grown from the sample. Third, it requires a skilled laboratory to conduct the test, thereby further increasing the cost to the patient. And finally, it requires that an uncomfortable, invasive procedure be performed on the patient to obtain the needed specimen. This method has a sensitivity of about eighty-five percent (85%) to ninety percent (90%).
(2) Chlamvdia
Chlamydia is caused by the bacterium Chlamydia trachomatous. If undetected and therefore untreated, chlamydia can cause pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy and infertility. In addition, undetected chlamydia is thought to cause about fifty percent (50%) of the nonspecific sexually transmitted infections, including nongonococcal urethritis and nonspecific urethritis. Each year, there are approximately four million (4,000,000) cases of chlamydia reported in the United States and seventy-two million (72,000,000) cases reported worldwide.
A standard method used in diagnosing chlamydia involves obtaining a scraping from the patient, which in women is taken from the endocervix. The scraping is then placed in a sterile nutritive medium and observed under a microscope for signs of microbial growth and the disease organism. A limitation on the use of this diagnostic method is the high cost, because a physician must procure the sample and skilled laboratory personnel must perform the test. Another limitation is the prolonged incubation time required to grow a culture. Also, it requires that an uncomfortable, invasive procedure be performed on the patient to gather the desired specimen. This method has a sensitivity of about seventy-five percent (75%) to eighty-five percent (85%).
The pathogenic microbial proteins that cause chlamydia may be detected in an endocervical scraping by direct fluorescent antibody testing (DFA) and enzyme-linked immunosorbent assay (ELISA), and by serological tests involving either complement fixation or microimmunifluorescence. These antigen detection tests for chlamydia are easily performed and are less costly than cultures. However, these methods are limited by a lower sensitivity than cultures and a low positive predictive value in low prevalence populations.
(3) Trichomoniasis
Trichomoniasis is caused by the flagellate protozoan Trichomonas vaginalis. If undetected and therefore untreated, trichomoniasis can cause vaginitis, urethritis, cystitis and prostatitis. Trichomoniasis afflicts approximately four million (4,000,000) people in the United States and one-hundred-eighty million (180,000,000) people worldwide. The "gold standard" test used to diagnose trichomoniasis is culture on a specimen taken from the posterior fornix. While this test is highly sensitive, its use is hindered by laboratory availability. Therefore, trichomoniasis is more commonly diagnosed by wet mount microscopy conducted on a similar specimen. The disadvantage of using wet mount microscopy to detect trichomoniasis is that this test has a low sensitivity of only about fifty percent (50%).
The newer techniques of PCR and LCR can detect sexually transmitted diseases by relying on nucleic acid amplification with subsequent detection. These tests are considered the most accurate diagnostic methods available. Nucleic acid amplification is presently commercially available for Chlamydia trachomatous and Neisseria gonorrhea and is under development for Trichomonas vaginalis. The important feature of these methods is their reliance on the mere presence of a small amount of the infectious organism, not on its viability. This reliance, on only the presence of the organism, makes it possible for the patient to self-collect a specimen such as urine and/or a swab from the vaginal introitus. The patient can then deposit the sample with a testing laboratory, by whatever means convenient, with no decrease in testing sensitivity. While these newer testing methods remove the requirement, and therefore the cost, of a physician being needed to obtain the sample, a major drawback associated with these methods is the increased cost of the diagnostic test itself.
Limitations in the above-mentioned methods of screening and diagnosing inflammatory conditions include; the inaccuracy of the methods, the need for a competent laboratory to perform the diagnostic test, delay between testing and results, and the necessity that a physician, possibly a high-priced specialist, be employed to collect the sample required for testing. All these factors combine to result in greater expense to the patient. Nowhere in the related art is there disclosed or suggested a method of screening for such inflammatory conditions which is relatively accurate, quick and inexpensive.
Therefore, there is a definite need for such a method of screening for inflammatory diseases such as an intraamniotic infection, bacterial meningitis, and the sexually transmitted diseases; gonorrhea, chlamydia and trichomoniasis, which is relatively accurate, quick, and economical, so that a greater number of people can be reliably and rapidly screened for these diseases. This screening would result in the appropriate diagnostic tests being utilized much more efficiently than is currently the case. Also, the patients thereby diagnosed could receive the correct medical treatment earlier in the pathology of the disease, when such intervention is likely to be more effective and less costly.
Neutrophils, or polymorphonuclear leukocytes, are specialized white blood cells, which are recruited to the site of a microbial invasion such as an infection. Once there, these neutrophils engulf and consume the invading microbes. The neutrophils digest the invaders by releasing toxic, antimicrobial peptides such as the defensins and lactoferrin into the phagosome. Defensins and lactoferrin are abundant in patients with inflammatory diseases such as an intraamniotic infection, bacterial meningitis and the sexually transmitted diseases; gonorrhea, chlamydia and trichomoniasis.
Defensins, also known as neutrophil peptides (NP), are a family of small, cationic, cysteine-rich antimicrobial peptides. In humans, they are designated "HNP." The predominant forms are HNP1, HNP2 and HNP3, which are herein referred to as the aggregate, "defensins HNP1-3." Defensins are found in the primary granules of the neutrophil. The defensins constitute greater than five percent (5%) of the total cellular protein and thirty percent (30%) to fifty percent (50%) of the total granule protein. Neutrophil defensins are highly stable to prolonged storage and are resistant to proteolysis and pH effects. The defensins increase membrane permeability by forming channels in the outer membranes of microbial cells and targeted host cells. Studies have shown that neutrophil defensins HNP1-3 are elevated in the blood plasma of patients with either bacterial meningitis or septicemia. This type of method is described in greater detail in, Journal of Laboratory and Clinical Medicine, 122: 202-207 (1993). Measuring the concentrations of other defensins such as HNP4, human defensins HD-5 and HD-6, and the human .beta. -defensins, HBD-1 and HBD-2, can also be done, either individually, or in combination with each other and/or the defensins HNP1-3, in accordance with the present invention.
Lactoferrin is an iron-binding, antimicrobial glycoprotein found in the secondary granules of the neutrophil and in endothelial tissues throughout the body. Like the defensins, lactoferrin is resistant to proteolysis and pH effects and can be stored for a prolonged period. Lactoferrin acts as a bacteriostatic agent by inhibiting bacterial growth. It does this by competing with bacteria for iron. Lactoferrin is also thought to possess a direct bactericidal activity that makes microbes more susceptible to attack by other antimicrobial peptides such as the defensins. Studies have shown that lactoferrin levels are elevated in the cerebrospinal fluid of patients with bacterial or viral meningitis. This type of method is described in more detail in, Acta Paediatrica Scandinavica 76: 987-988 (1987).
The present invention provides a method of evaluating, i.e., diagnosing and/or screening a patient for inflammatory diseases such as an intraamniotic infection, bacterial meningitis and the sexually transmitted diseases; gonorrhea, chlamydia and trichomoniasis. The present invention accomplishes this screening by measuring the concentration of neutrophil defensins, preferably neutrophil defensins HNP1-3, and the concentration of lactoferrin, found in a bodily fluid, a tissue or a combination thereof, adding these two concentrations together to yield a summed total or creating a threshold value of a dipstick test which both lactoferrin and defensins must meet. This measured sum total or ascertainment of a threshold value will give an indication of whether the patient is at risk of suffering from inflammatory diseases such as an intraamniotic infection, bacterial meningitis or the sexually transmitted diseases; gonorrhea, chlamydia and trichomoniasis. Such a screening method, as is herein provided, has never been disclosed or suggested in the prior art. The advantages and benefits of the present invention will be apparent from the Detailed Description of the Invention herein below.